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• Microscopic Examination of urine is used to detect and identify insoluble materials in urine
• Microscopic examination of urine must include quantitation of elements present
• Some elements are considered normal unless present in increased amounts
• Microscopic examination of urine is the least standardized and most consuming type of routine urinalysis
• What the Urine Microscopist must know

  • Clinical relevance of urine findings
  • Chemical abnormalities associated with microscopic interpretations
• Formed Elements found in urine

  • Cellular elements
  • Casts
  • Crystals
  • Organisms (Bacteria, Fungi, Viral inclusion cells, Parasites)
• Abnormalities in the physical and chemical portions of urinalysis play a primary role in the decision to perform a microscopic analysis
• Microscopic examination is not followed in the Philippines
• CLSI recommends that microscopic examination be performed when

  • Requested by a physician
  • A laboratory-specified population is being tested
  • Any abnormal or physical or chemical result is obtained
• Macroscopic Screening Correlations


  • Blood (Color, Clarity, RBCs/RBC casts)
  • Protein (Casts/Cells, Bacteria/WBCs)
  • Nitrite (WBCs/WBC casts/bacteria)
  • Leukocyte Esterase (WBCs)
  • Glucose (Yeast)
• Factors causing variations in Urine Microscopic Analysis

  • Methods by which the sediment is prepared
  • Volume of sediment examined
  • Methods and equipment used to obtain visualization
  • Manner in which results are reported
• Conventional method


  1. Centrifuge urine

  2. Transfer to glass slide

  3. Add cover slip

  4. Examine under the microscope
• Quantitative Counts


  • Uses the hematocytometer
  • Uses undiluted well-mixed urine
• Normal values: neutrophils = 5–30/μL, RBCs = 3–20/μL, casts = 1–2/μL
• Kesson (1978) provided evidence that chamber counts on centrifuged urine sediments are more reliable in predicting renal functional abnormalities than is a conventional method using cells per HPF
• Addis Count


  1. Developed by Addis in 1926

  2. Uses a hematocytometer to count the number of RBCs, WBCs, casts, and epithelial cells

  3. Specimen: 12 hour urine
• Normal values per 12 hours: RBCs = 0-500,000, WBCs & epithelial cells= 0-1,800,000, Hyaline cast = 0-5,000
• Commercial Systems


  • KOVA (Hycor Biochemical, Inc.)
  • Urisystem (ThermoFisher Scientific)
  • Count-10 (V-Tech, Inc.)
  • Quick-Prep Urinalysis System (Global Scientific)
  • Censlide 2000 Urinalysis System (International Remote Imaging Systems)
  • R/S Workstations 1000, 2000, 2003 (DioSys)
  • IRIS Urinalysis Workstation
• Specimen Preparation


  • Specimens should be examined while fresh
  • Refrigeration may cause precipitation of crystals (Warm specimen to 37ºC to dissolve!)
  • Thoroughly mix the specimen prior to decanting a portion into a centrifuge tube
• Routine Urine Analysis - Specimen Volume

  • Standard amount centrifuge usually 10-15 mL
  • 12 mL frequently used
  • If obtaining a 12 mL specimen is not possible: Specimen volume noted on report, For correction: Example: if only 6 mL of urine is centrifuged, results are multiplied by 2
• Routine Urine Analysis - Centrifugation


  • 5 minutes @ 400 RCF (RCF = 1.118 X 10-5 X radius in cm X RPM2)
  • Braking mechanism to slow down centrifuge should not be used
  • All specimens must be centrifuged in capped tubes
• Routine Urine Analysis - Sediment Preparation


  • Concentration factor = volume of urine centrifuged ÷ sediment volume (used when quantitating the number of elements per mL)
  • Sediment volume frequently used are 0.5 and 1.0 mL
  • Resuspend urine sediment in tube prior to microscopic examination (by pipette or by tapping the tube with the finger, avoid vigorous agitation)
  • Urine should be aspirated rather than poured off
• Routine Urine Analysis - Examination of the Sediment

  • Volume of sediment examined: Glass slide method: 20uL covered by a 22 x 22 mm cover slip, Commercial systems: control volume of sediment by providing slides with chambers
  • Urine sediment examined in a minimum of 10 LPO and 10 HPO, LPO scanning of cover-slip perimeter is recommended for detection of casts
  • Use reduced light when examining unstained sediment by bright-field microscope
• Reporting the Microscopic Examination

  • RBCs/hpf (Grade 0, 1+, 2+, 3+, 4+)
  • WBCs/hpf (Grade 0, 1+, 2+, 3+, 4+)
  • Casts/lpf (Hyaline, granular, fine, coarse, waxy, cellular, rbc, wbc, etc, Grade 0, 1+, 2+, 3+, 4+)
  • Normal crystals/lpf (Acid urine: Amorphous urates, Uric acid, Calcium Oxalates, Alkaline urine: Amorphous phosphate, Triple phosphate, Grade None, Occasional, Few, Many)
  • Miscellaneous structures/lpf (Squamous epithelial cells, Round epithelial cells, Bacteria, Yeast, Sperm cells, Mucus threads, parasites, others, Grade None, Occasional, Few, Many)
• Conversion of the number of elements per lpf or hpf to the number per mL provides standardization
• Sediment stains


  • Sternheimer-Malbin stain
  • 0.5% toluidine blue
  • 2% acetic acid
  • Lipid stains (Oil red O and Sudan III)
  • Gram stain
  • Hansel stain
  • Prussian Blue stain
• Cytodiagnostic Urine Testing is not part of Routine urinalysis
• Cytodiagnostic Urine Testing


  • Slides prepared by cytocentrifugation and stained with Papanicolaou stain
  • Performed by the cytology laboratory
  • Purpose: For detection of malignancies of the lower urinary tract, To provide definitive information about renal tubular changes associated with transplant rejection; viral, fungal, and parasitic infections; cellular inclusions; pathologic casts; and inflammatory conditions
• Essential parts of a Microscope

  • Lens system (Oculars, Objectives, Coarse- and fine-adjustment knobs)
  • Illumination system (Light source, Condenser, Field and iris diaphragm)
  • Body (Base, Body tube, Nosepiece)
• Illumination system


  • Light source (Lamps/bulbs: Tungsten, Halogen, Fluorescent, LED, Regulated by a Rheostat, Filters providing enhanced contrast and color correction)
  • Condenser (gather the light coming from the light source and to concentrate that light in a collection of parallel beams into the specimen)
  • Field and iris (aperture) diaphragm (Iris Diaphragm controls the amount of light passing through the slide or specimen, Field Diaphragm may also be present in the light source; may be opened or closed; used in focusing the light which passes up through the condenser)
• Lens system


  • Objectives (Perform the initial magnification, Inscriptions: Objective magnification, Total magnification, thickness of the cover glass, labeled OIL or HI or OEL, numerical aperture)
  • Oculars/Eyepiece lens (Responsible for further resolution)
  • Coarse- and fine-adjustment knobs
• Kohler Illumination


  1. Adjustments done to provide optimal viewing of illuminated objects

  2. Steps: Place a slide on the stage and focus using LPO with the condensers raised, Close the field diaphragm, Lower he condenser until the edges of the field diaphragm are sharply focused, Center the image of the field diaphragm with the condenser centering screws, Open the field diaphragm until its image is at the edge of the field, Remove an eyepiece and look down through the eyepiece tube, Adjust the aperture diaphragm until approximately 75% of the field is visible, Replace the eyepiece
• The image in the microscope is upside down and reversed
• Types of Microscope


  • Brightfield
  • Phase contrast
  • Polarizing
  • Dark field
  • Fluorescence
  • Interference contrast
• Brightfield Microscopy


  • Most common type of microscopy performed in the urinalysis laboratory
  • Object appear dark against a light background
  • Sediments must be examined using decreased light controlled by adjusting the rheostat
  • Sediment constituents with a low refractive index will be overlooked when subjected to light with high intensity
• Phase Contrast Microscopy


  • Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucus threads, and Trichomonas
  • Eliminates the need to fix or stain living cells
  • Accomplished by adaptation of a bright-field microscope
  • Phase-contrast objective lens – contains a phase ring that that retards light
  • As light rays are slowed, there is a decrease in the intensity of light producing contrast
  • Specific condenser – contains also a phase ring with a central clear circular area
• Polarizing Microscope


  • The use of polarized light aids in identification of birefringent elements: cholesterol on OFB, fatty casts, and crystals
  • Birefringent – a property indicating that the element can refract light in 2 dimensions at 90 degrees to each other
  • Obtaining a polarized light: Brightfield microscopes could be used, 2 filters installed in a cross formation: 1st filter - Polarizing filter, 2nd filter - Analyzer
  • Interpretation: Nonbirefringent objects = no refracted light = no light reaches analyzer = object appears black, Birefringent objects = refracted light = refracted light reaches analyzer = object appears white/colored against a black background
• Interference-Contrast Microscopy


  • Produces a 3-dimensional microscopy-image and layer-by-layer imaging of a specimen
  • Split the light ray so that the beams pass through different areas of the specimen. The light interference produced by the varied depths of the specimen generates a 3-dimensional image
  • Object appears bright against a dark background
  • 2 types: Modulation contrast (Hoffman), Differential-interference contrast (Nomarski)
• Dark-Field Microscopy


  • Used to enhance visualization of specimens not viewed with a bright-field microscope
  • Aids in identification of Treponema pallidum
  • A bright-field microscope is adapted, Replacing the condenser with a dark-field condenser that contains an opaque disk, Disk blocks light from directly entering the objective, Light rays pass around the specimen and are scattered, producing a bright image on a dark background
• Polarizing microscopy


  Used in urinalysis to confirm the identification of fat droplets, OFB, and fatty casts that produces a characteristic maltese cross pattern